1. Copyright © 2007 Asuragen, Inc NAT Testing in Solid Tissues and Organs: A Pre-analytical Solution for Nucleic Acid Stabilization Cindy WalkerPeach, Ph.D. cwalkerpeach@asuragen.com

2. Copyright © 2007 Asuragen, Inc Ambion Diagnostics Becomes Asuragen! Ambion, Inc. Formed 1989 Ambion Research Products Division Ambion Diagnostics Division Formed 2000 Ambion Services Division Formed 2005 Purchased by Applied Biosystems Group (NYSE: ABI) for $273M March 2, 2006 Asuragen, Inc. Ambion Diagnostics Ambion Services Key Ambion R&D Scientists and Executive Team

3. Copyright © 2007 Asuragen, Inc RNARetain ™ Pre-analytical Solution Overview Applications Examples Conclusions

4. Copyright © 2007 Asuragen, Inc RNARetain Overview Versatile, non-toxic, non-organic solution for preservation of nucleic acids by inactivating nucleases to prevent degradation of RNA/DNA Stabilizes nucleic acids at room temperature, without requirement of dry ice or liquid nitrogen, thereby allowing biological samples to be shipped on wet ice or at ambient temperatures Compatible with all downstream RNA extraction methods Based on formulation widely used in life science research (RNAlater - Ambion, Inc.) Covered by US patent: 6,204,375 and WW equivalents

5. Copyright © 2007 Asuragen, Inc RNARetain - cGMP Traceability Manufactured under QSR 21 CFR Part 820 guidelines to ensure lot-to-lot consistency critical for reagents used in clinical specimen collection and storage for downstream RNA extraction and molecular analysis Compatibility with major extraction procedures and molecular analysis techniques will allow end-user to qualify reagent for use in specific downstream applications cGMP traceability provides consistent performance in end- user’s hands

6. Copyright © 2007 Asuragen, Inc Selected Publications Development and validation of a method for using breast core needle biopsies for gene expression microrarray analyses Matthew Ellis, et al. Clin Cancer Res. 2002, 8:1155-1166 The effect of sample degradation and RNA stabilization on classical swine fever virus RT-PCR and ELISA methods Stuart D. Blacksell, Syseng Khounsy and Harvey A. Westbury. J. Vir Methods. 2004, 118(1):33-37 Viral infectivity is maintained by an RNA protection buffer C. Uhlenhaut and M. Kracht. J. Virol Methods. 2005, 128(1-2):189-91 Prognostic gene expression signatures can be measured in tissues collected in RNAlater preservative Dondapati Chowdary, et al. JMD. 2006, Vol. 8, No. 1 Currently there are over 1,000 citations since Jan 2001 in HighWire Press: http://Highwire.Stanford.eduhttp://Highwire.Stanford.edu

7. Copyright © 2007 Asuragen, Inc Unmet Clinical Need………… Clinical Research, Tissue/Cell Banking, Expression Profiling Services, Molecular Diagnostics Molecular Isolation (RNA/DNA) “Freezes” mRNA and microRNA Expression Profiles Clinical Easy and Safe Sample Acquisition Convenient Sample Transport Virus Preservation Long-Term Cell/ Tissue Banking

8. Copyright © 2007 Asuragen, Inc Intracellular RNA Preservation 5E+5 MCF-7 Breast Tissue Cells mirVana ™ or Trizol ® Extraction Flash Freeze Or RNARetain Nanodrop ® Agilent Bioanalyzer x4 RNARetainFlash Freeze mirVana Trizol RNARetain preserves the quality of intracellular RNA to the same extent as the currently established standard of flash-freezing

9. Copyright © 2007 Asuragen, Inc qRT-PCR Reproducibility 5E+5 MCF-7 Cells In RNARetain mirVana Extraction 1E+3 HCV1b ARQ Process Control HCV1b (process con) and TBP (internal con) qRT-PCR at 40ng sample input x5 x3 Reproducible RNA quality and qRT-PCR amplification efficiency is achieved for intracellular RNA extracted from RNARetain preserved cells

10. Copyright © 2007 Asuragen, Inc Armored RNA ® Recovery from RNARetain 1E+7 HCV1b ARQ + or - RNARetain RNA Isolation (Trizol or mirVana Kit) HCV1b qRT-PCR x3 + - + - RNARetain mirVanaTrizol Pseudo-viral particles can be preserved in and extracted from RNARetain in a reproducible manner

11. Copyright © 2007 Asuragen, Inc RNARetain Data Summary Preserves quality of intracellular RNA to same extent as flash-freezing Does not interfere with reproducibility of intracellular RNA extraction and analysis methods Effective for storage and preservation of viral RNA as indicated by compatibility with Armored RNA

12. Copyright © 2007 Asuragen, Inc C Combination of two microRNAs can be used to distinguish normal, chronic pancreatitis, and carcinoma samples Case Study:

13. Copyright © 2007 Asuragen, Inc Analysis of FNA Specimens Endoscopic microdissection of pancreatic mass (EU-FNA) Preservation in RNARetain RNA Isolation microRNA expression profiling (qRT-PCR) n=7n=2 n=10n=7 Frozen tissues from Szafranska et al. Frozen Signal ratios for two miRNAs showing differential expression specific to cancerous (ca) or normal (N) tissue are maintained in RNARetain (FNA samples) RNARetain preserves miRNA signatures for downstream molecular profiling Frozen

14. Copyright © 2007 Asuragen, Inc Closing Remarks RNARetain is a pre-analytical solution which stabilizes intracellular RNA in cells and solid tissues for downstream extraction and molecular characterization RNARetain is compatible with Armored RNA, illustrating potential utility in collection and storage of viral-infected tissues, thus eliminating the need for immediate processing or flash freezing Next step will be to identify beta test sites to evaluate clinical utility of RNARetain for collection and transport of viral-infected tissue specimens Agendia, B.V. Used in conjunction with MammaPrint® 70 gene expression signature which predicts the risk of breast cancer recurrence Received US FDA approval Feb 6, 2007